Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States.
Department of Chemistry and Biochemistry and the Viral Information Institute, San Diego State University, San Diego, California 92182, United States.
J Am Chem Soc. 2022 Aug 17;144(32):14647-14656. doi: 10.1021/jacs.2c04142. Epub 2022 Aug 5.
Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems; however, approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO treatment. Furthermore, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.
荧光成像是探测生物系统中大分子动力学的一种强大方法；然而，用于细胞 RNA 成像的方法仅限于单个 RNA 结构的研究或主要与固定样本兼容的批量 RNA 标记方法。在这里，我们开发了一种用于在活细胞中进行批量 RNA 动力学荧光成像的平台。我们表明，通过过量表达尿苷-胞苷激酶 2，荧光双环和三环胞苷类似物可以被代谢掺入细胞 RNA 中。特别是，用三环胞苷衍生的核苷 tC 进行代谢喂养，结合共聚焦成像，可以在单细胞分辨率下研究 RNA 的合成、降解和运输。我们将我们的成像方式应用于研究氧化应激反应过程中的 RNA 代谢和定位，并发现 NaAsO 处理后大量 RNA 周转大大加速。此外，我们鉴定了含有氧化应激过程中产生的 RNA 转录本的细胞质 RNA 颗粒，这些颗粒与经典应激颗粒和 P 体不同，并且与 RNA 解旋酶 DDX6 共定位。总之，我们的工作为活细胞 RNA 成像提供了一种强大的方法，并揭示了细胞如何响应氧化应激重塑 RNA 转录组动力学。
